Molecular and functional characteristics of ovarian surface epithelial cells transformed by KrasG12D and loss of Pten in a mouse model in vivo

Abstract

Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage. Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear. We recently generated Pten(fl/fl); Kras(G12D); Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of Kras(G12D) in ovarian surface epithelial (OSE) cells. On the basis of histopathologic criteria, the mutant mice developed low-grade ovarian serous papillary adenocarcinomas at an early age and with 100% penetrance. This highly reproducible phenotype provides the first mouse model in which to study this ovarian cancer subtype. OSE cells isolated from ovaries of mutant mice at 5 and 10 weeks of age exhibit temporal changes in the expression of specific Mullerian epithelial marker genes, grow in soft agar and develop ectopic invasive tumors in recipient mice, indicating that the cells are transformed. Gene profiling identified specific mRNAs and microRNAs differentially expressed in purified OSE cells derived from tumors of the mutant mice compared with wild-type OSE cells. Mapping of transcripts or genes between the mouse OSE mutant data sets, the Kras signature from human cancer cell lines and the human ovarian tumor array data sets, documented significant overlap, indicating that KRAS is a key driver of OSE transformation in this context. Two key hallmarks of the mutant OSE cells in these mice are the elevated expression of the tumor-suppressor Trp53 (p53) and its microRNA target, miR-34a-c. We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

Figure 1

Temporal changes in OSE cell morphology and expression of specific genes associated with the serous adenocarcinoma phenotype in the Pten;Kras^G12D;Amhr2-cre mice. A.) H&E and immuno-labeling of ovarian sections from WT and mutant mice at 5 and 10 weeks of age. B.) Gene expression patterns in whole ovaries of WT or tumor bearing (T) Pten;Kras^G12D;Amhr2-cre mice at 5 and 10 weeks of age.

Figure 2

Genes overlapping between the Pten;Kras;Amhr2-cre gene signature and relevant signatures from human cancers. (A) Overlaps between the set of human orthologs high (fold>2) in Pten;Kras;Amhr2-cre mice (compared to wild-type mice) and various sets of genes derived from human datasets: genes high/low (p<0.01, t-test) in k-ras mutant versus k-ras wild-type cell lines, and genes high/low (p<0.05) in serous, mucinous, or endometrioid ovarian cancers, as compared to normal ovary. Numbers of genes in each set indicated in parentheses. Numbers overlapping between sets were compared to chance expected. (B) Heat maps showing expression patterns for the Pten;Kras;Amhr2-cre gene signature, both in the Pten;Kras;Amhr2-cre mouse expression dataset and in an expression dataset of human cancer cell lines with or without mutations in kras (35). (C) Heat maps for the Pten;Kras;Amhr2-cre gene signature, both in the mouse dataset and in a dataset of human ovarian tumors (36). For parts B and C, genes represented in both heat maps are the same and have the same ordering, and genes not represented in the given human dataset are not shown.

Figure 3

Mutant OSE cells express genes associated with human serous adenocarcinomas in a time-dependent manner. A.) Cytokeratin 8 and E-cadherin-positive OSE cells removed from the ovarian surface exhibit a cobblestone appearance. B.) Expression of genes in RNA samples from whole ovaries and purified OSE cells isolated from ovaries of wild type and Pten;Kras^G12D;Amhr2-cre mice at 5 and 10 weeks of age.

Figure 4

OSE cells isolated from ovaries of Pten;Kras;Amhr2-cre mice are transformed. A.) Mutant OSE cells proliferate faster than WT cells at 24 and 48h. B.) Mutant but not WT OSE cells grow in soft agar (C) and form ectopic tumors in the peritoneal cavity, subcutaneously and under the kidney capsule in vivo (D).

Figure 5

The MEK1/ERK1/2 and PI3K/AKT pathways drive gene expression and transformation of the mutant OSE cells. A.) Western blot of lysates prepared from mutant OSE cells cultured in media alone or with either the MEK1 inhibitor UO126 (10 □M) or the PI3K inhibitor LY294002 (10 □M) for 24 hrs. B.) Colony formation of mutant OSE cells grown in soft agar is inhibited by both UO126 and LY294002. C.) Gene expression is altered in cells cultured in media alone or with either UO126 or LY294002 for 24h. D.) TRP53 immunolabeling decreases in mutant cells cultured for 24 hours with or without 10 □M UO126 or LY294002 compared to media alone.

Figure 6

Mutations in either Pten or Kras alone alter gene expression in purified OSE cells. A.) OSE cells were isolated from Pten;Amhr2-cre, Kras;Amhr2-cre, Pten;Kras;Amhr2-cre (tumor-bearing, T) and WT mice. The transformed cells (T) grow rapidly and eventually form clusters whereas the WT and KRAS OSE cells exhibit slower growth and well-defined boarders. OSE cells from the Pten null cells, like the transformed (T) cells, lack distinct borders but are not transformed. B.) The transformed cells and the Pten null cells exhibit striking similarities in the expression of specific mRNAs, including Trp53 and its target p21 (Cdkna1).

Figure 7

Specific miRNAs are expressed in the mutant compared to WT OSE cells and are regulated by inhibitors of MEK1/ERK1/2 and PI3K as shown by real-time RT-PCR. A and B.) Primary miRNA and mature transcripts are transcriptionally regulated in mutant OSE cells at 5 and 10 weeks of age. C.) Transcription of primary miRNAs is altered by UO126 or LY294002. D.) miR-34a is elevated in OSE cells from Pten;Kras;Amhr2-cre (tumor-bearing, T) mice and from Pten;Kras;Amhr2-cre mice (Pten) but not from Kras;Amhr2-cre mice (Kras). Conversely, the potential tumor suppressor miR-31 is down-regulated in the tumor OSE cells and Kras cells but not the Pten null cells, indicating differential regulation of these two pathways.